Aging Yeast Cells Research Articles

Noise reduction as an emergent property of single-cell aging

Noise-induced heterogeneity in gene expression is an inherent reality for cells. However, it is not well understood how noise strength changes for a single gene while the host cell is aging. Using a state-of-the-art microfluidic platform, we measure noise dynamics in aging yeast cells by tracking the generation-specific activity of the canonical GAL1 promoter. We observe noise reduction during normal aging of a cell, followed by a short catastrophe phase in which noise increased. We hypothesize that aging-associated increases in chromatin state transitions are behind the observed noise reduction and a stochastic model provides quantitative support to the proposed mechanism. Noise trends measured from strains with altered GAL1 promoter dynamics (constitutively active, synthetic with nucleosome-disfavoring sequences, and in the absence of RPD3, a global remodeling regulator) lend further support to our hypothesis. Observing similar noise dynamics from a different promoter (HHF2) provides support to the generality of our findings.

Sphingolipids facilitate age asymmetry of membrane proteins in dividing yeast cells.

One proposed mechanism of cellular aging is the gradual loss of certain cellular components that are insufficiently renewed. In an earlier study, multidrug resistance transporters (MDRs) were postulated to be such aging determinants during the yeast replicative life span (RLS). Aged MDR proteins were asymmetrically retained by the aging mother cell and did not diffuse freely into the bud, whereas newly synthesized MDR proteins were thought to be deposited mostly in the bud before cytokinesis. In this study, we further demonstrate the proposed age asymmetry of MDR proteins in dividing yeast cells and investigate the mechanism that controls diffusive properties of MDR proteins to maintain this asymmetry. We found that long-chain sphingolipids, but not the septin/endoplasmic reticulum-based membrane diffusion barrier, are important for restricting MDR diffusion. Depletion of sphingolipids or shortening of their long acyl chains resulted in an increase in the lateral mobility of MDR proteins, causing aged MDR protein in the mother cell to enter the bud. We used a mathematical model to understand the effect of diminished MDR age asymmetry on yeast cell aging, the result of which was qualitatively consistent with the observed RLS shortening in sphingolipid mutants.

Increased genome instability is not accompanied by sensitivity to DNA damaging agents in aged yeast cells

The budding yeast Saccharomyces cerevisiae divides asymmetrically, producing a new daughter cell from the original mother cell. While daughter cells are born with a full lifespan, a mother cell ages with each cell division and can only generate on average 25 daughter cells before dying. Aged yeast cells exhibit genomic instability, which is also a hallmark of human aging. However, it is unclear how this genomic instability contributes to aging. To shed light on this issue, we investigated endogenous DNA damage in S. cerevisiae during replicative aging and tested for age-dependent sensitivity to exogenous DNA damaging agents. Using live-cell imaging in a microfluidic device, we show that aging yeast cells display an increase in spontaneous Rad52 foci, a marker of endogenous DNA damage. Strikingly, this elevated DNA damage is not accompanied by increased sensitivity of aged yeast cells to genotoxic agents nor by global changes in the proteome or transcriptome that would indicate a specific “DNA damage signature”. These results indicate that DNA repair proficiency is not compromised in aged yeast cells, suggesting that yeast replicative aging and age-associated genomic instability is likely not a consequence of an inability to repair DNA damage.

Papers of note in Science 355 (6330)

This week’s articles include several reviews on targeting signaling pathways to treat cancer, as well as research articles that highlight proteins that drive circadian clocks; how bacteriophages affect the virulence of pathogenic bacteria; a mobile transcription factor in plants; a secreted nucleoside that affects metabolism; and the effects of protein aggregation in aging yeast cells.

Old moms say, no Sir.

Asymmetric distribution of an aggregated protein controls yeast cell aging

The oxidation state of the cytoplasmic glutathione redox system does not correlate with replicative lifespan in yeast.

What is cause and what is consequence of aging and whether reactive oxygen species (ROS) contribute to this phenomenon is debated since more than 50 years. Notwithstanding, little is known about the cellular buffer and redox systems in aging Saccharomyces cerevisiae, which is a model for aging stem cells. Using genetically encoded fluorescent sensors, we measured pH, H2O2 levels and the glutathione redox potential compartment-specific in the cytosol of living, replicatively aging yeast cells, growing under fermenting and respiratory conditions until the end of their lifespan. We found that the pH decreases under both conditions at later stages of the replicative lifespan. H2O2 levels increase in fermenting cells in the post-replicative stage, but increase continuously with age in respiring cells. The glutathione redox couple becomes also more oxidizing in respiring cells but surprisingly more reducing under fermenting conditions. In strains deleted for the gene encoding glutathione reductase Glr1, such a reduction of the glutathione redox couple with age is not observed. We demonstrate that in vivo Glr1 is activated at lower pH explaining the reduced glutathione potential. The deletion of glr1 dramatically increases the glutathione redox potential especially under respiratory conditions but does not reduce lifespan. Our data demonstrate that pH and the glutathione redox couple is linked through Glr1 and that yeast cells can cope with a high glutathione redox potential without impact on longevity. Our data further suggest that a breakdown of cellular energy metabolism marks the end of replicative lifespan in yeast.

Absence of Non-histone Protein Complexes at Natural Chromosomal Pause Sites Results in Reduced Replication Pausing in Aging Yeast Cells

There is substantial evidence that genomic instability increases during aging. Replication pausing (and stalling) at difficult-to-replicate chromosomal sites may induce genomic instability. Interestingly, in aging yeast cells, we observed reduced replication pausing at various natural replication pause sites (RPSs) in ribosomal DNA (rDNA) and non-rDNA locations (e.g., silent replication origins and tRNA genes). The reduced pausing occurs independent of the DNA helicase Rrm3p, which facilitates replication past these non-histone protein-complex-bound RPSs, and is independent of the deacetylase Sir2p. Conditions of caloric restriction (CR), which extend life span, also cause reduced replication pausing at the 5S rDNA and at tRNA genes. In aged and CR cells, the RPSs are less occupied by their specific non-histone protein complexes (e.g., the preinitiation complex TFIIIC), likely because members of these complexes have primarily cytosolic localization. These conditions may lead to reduced replication pausing and may lower replication stress at these sites during aging.

Caloric restriction alleviates alpha-synuclein toxicity in aged yeast cells by controlling the opposite roles of Tor1 and Sir2 on autophagy

Alpha-synuclein (syn) is the main component of proteinaceous inclusions known as Lewy bodies (LBs), which are implicated in the pathogenesis of the neurodegenerative diseases known as synucleinopathies, like Parkinson’s disease (PD). Aging is a major risk factor for PD and thus, interventions that delay aging will have promising effects in PD and other synucleinopathies. Caloric restriction (CR) is the only non-genetic intervention shown to promote lifespan extension in several model organisms. CR has been shown to alleviate syn toxicity and herein we confirmed the same effect on the yeast model for synucleinopathies during chronological lifespan. The data gathered showed that TOR1 deletion also results in similar longevity extension and abrogation of syn toxicity. Intriguingly, these interventions were associated with decreased autophagy, which was maintained at homeostatic levels. Autophagy maintenance at homeostatic levels promoted by CR or TOR1 abrogation in syn-expressing cells was achieved by decreasing Sir2 levels and activity. Furthermore, the opposite function of Tor1 and Sir2 in autophagy is probably associated with the maintenance of autophagy activity at homeostatic levels, a central event linked to abrogation of syn toxicity promoted by CR.

Yeast Replicator: A High-Throughput Multiplexed Microfluidics Platform for Automated Measurements of Single-Cell Aging.

The yeast Saccharomyces cerevisiae is a model organism for replicative aging studies; however, conventional lifespan measurement platforms have several limitations. Here, we present a microfluidics platform that facilitates simultaneous lifespan and gene expression measurements of aging yeast cells. Our multiplexed high-throughput platform offers the capability to perform independent lifespan experiments using different yeast strains or growth media. Using this platform in minimal media environments containing glucose, we measured the full lifespan of individual yeast cells in wild-type and canonical gene deletion backgrounds. Compared to glucose, in galactose we observed a 16.8% decrease in replicative lifespan accompanied by an ∼2-fold increase in single-cell oxidative stress levels reported by PSOD1-mCherry. Using PGAL1-YFP to measure the activity of the bistable galactose network, we saw that OFF and ON cells are similar in their lifespan. Our work shows that aging cells are committed to a single phenotypic state throughout their lifespan.

Effects of Selenium on Morphological Changes in Candida utilis ATCC 9950 Yeast Cells.

This paper presents the results of microscopic examinations of the yeast cells cultured in yeast extract–peptone–dextrose (YPD) media supplemented with sodium selenite(IV). The analysis of the morphological changes in yeast cells aimed to determine whether the selected selenium doses and culturing time may affect this element accumulation in yeast cell structures in a form of inorganic or organic compounds, as a result of detoxification processes. The range of characteristic morphological changes in yeasts cultivated in experimental media with sodium selenite(IV) was observed, including cell shrinkage and cytoplasm thickening of the changes within vacuole structure. The processes of vacuole disintegration were observed in aging yeast cells in culturing medium, which may indicate the presence of so-called ghost cells lacking intracellular organelles The changes occurring in the morphology of yeasts cultured in media supplemented with sodium selenite were typical for stationary phase of yeast growth. From detailed microscopic observations, larger surface area of the cell (6.03 μm2) and yeast vacuole (2.17 μm2) were noticed after 24-h culturing in the medium with selenium of 20 mg Se4+/L. The coefficient of shape of the yeast cells cultured in media enriched with sodium selenite as well as in the control YPD medium ranged from 1.02 to 1.22. Elongation of cultivation time (up to 48 and 72 h) in the media supplemented with sodium selenite caused a reduction in the surface area of the yeast cell and vacuole due to detoxification processes.

Aging Yeast Cells Are Fat!

Saccharomyces cerevisiae is an excellent model organism to understand the molecular mechanism of aging. Here we have used yeast haploid RAD deletion strains to explore the relationship between aging and lipid storage. RAD genes are mainly involved in DNA repair mechanism and hence, their deletions result in shorter life span. Viable rad mutants were screened for neutral lipid content and some of the mutants showed significantly high amount of triacylglycerol and steryl ester, besides short chronological life span. Of these, RAD50, MRE11 and XRS2 form a complex denoted as MRX which is involved in homologous recombination and hence the mutant cells are hypersensitive to DNA damage. Deletion of any one of the three genes in MRX complex abolished the function of the complex and exhibited the same phenotype. Microarray data of single MRX deletions revealed that besides DNA damage signature genes, lipid metabolism genes were also up‐regulated. We observed that rad50Δ, mre11Δ, xrs2Δ and mrxΔ have more number of lipid droplets (LDs) at stationary phase compared to wild type yeast cells. These mutants also showed a peculiar phenotype as observed under a scanning electron microscope and had short chronological life span compared to wild‐type. Since this aging phenotype correlated directly with the increase in number and size of lipid droplets, lipid storage could be a possible indicator of aging cells. Further, this phenomenon was also observed in normal wild type aging cells grown for over a month, with huge lipid droplets of ~2.0 µm in diameter. While lipid accumulation in stressed conditions has been reported, our findings demonstrate that aging cells accumulate neutral lipids.

Inhibition of free radical scavenging enzymes affects mitochondrial membrane permeability transition during growth and aging of yeast cells.

In this study, we investigated the change in the antioxidant enzymes activity, cell respiration, reactive oxygen species (ROS), and impairment of membrane mitochondria permeability in the Endomyces magnusii yeasts during culture growth and aging. We showed that the transition into stationary phase is the key tool to understanding interaction of these processes. This growth stage is distinguished by two-fold increase in ROS production and respiration rate as compared to those in the logarithmic phase. It results in induction of alternative oxidase (AO) in the stationary phase, decline of the main antioxidant enzymes activities, ROS-production, and mitochondria membrane permeability. Significant increase in the share of mitochondrial isoform of superoxide dismutase (SOD2) occurred in the stationary phase from 51.8% (24 h of cultivation) to 68.6% (48 h of cultivation). Upon blocking the essential ROS-scavenging enzymes, SODs and catalases (CATs) some heterogeneity of cell population was observed: 80-90% of cells displayed evident signs of early apoptosis (such as disorientation of mitochondria cristae, mitochondrial fragmentation and deformation of nuclear chromatine). However, 10-20% of the population were definitely healthy. It allowed to draw the conclusion that a complete system of cell antioxidant protection underlies normal mitochondria functioning while the E. magnusii yeasts grow and age. Moreover, this system provides unimpaired cell physiology under oxidative stress during culture aging in the stationary phase. Failures in mitochondria functions due to inhibition of ROS-scavenging enzymes of CATs and SODs could lead to damage of the cells and some signs of early apoptosis.

Yeast cell aging and death

Why study cell aging and cell death, in yeast? Most of us have heard these questions before, sometimes from fellow colleagues. Reports from National Institute of Aging (National Institutes of Health), the World Health Organization, and other relevant organizations, outline the substantial challenges related to aging that we will face in the next decade. This concern is also reflected in Horizon2020, the next EU framework for research and innovation, where the challenges of an aging population and the consequences it brings for society, are among the top priorities. One of the key areas of concern is the economic and societal impact of age-related …

Aging Yeast Cells Undergo a Sharp Entry into Senescence Unrelated to the Loss of Mitochondrial Membrane Potential

In budding yeast, a mother cell can produce a finite number of daughter cells before it stops dividing and dies. Such entry into senescence is thought to result from a progressive decline in physiological function, including a loss of mitochondrial membrane potential (ΔΨ). Here, we developed a microfluidic device to monitor the dynamics of cell division and ΔΨ in real time at single-cell resolution. We show that cells do not enter senescence gradually but rather undergo an abrupt transition to a slowly dividing state. Moreover, we demonstrate that the decline in ΔΨ, which is observed only in a fraction of cells, is not responsible for entry into senescence. Rather, the loss of ΔΨ is an age-independent and heritable process that leads to clonal senescence and is therefore incompatible with daughter cell rejuvenation. These results emphasize the importance of quantitative single-cell measurements to decipher the causes of cellular aging.

Approaches to study yeast cell aging and death

For millennia, yeast has been exploited to obtain fermentation products, such as foods and beverages. For c. 50years, yeast has been an established model organism for basic and applied research, and more specifically, for c. 15years, this unicellular organism has been applied to dissect molecular mechanisms of cell aging and programmed cell death. In this review, we present an overview of approaches to study cell aging and death in yeast, including lifespan assessments, calorie restriction, cell viability, survival, and death markers.

Aging and differentiation in yeast populations: elders with different properties and functions

Over the past decade, it has become evident that similarly to cells forming metazoan tissues, yeast cells have the ability to differentiate and form specialized cell types. Examples of yeast cellular differentiation have been identified both in yeast liquid cultures and within multicellular structures occupying solid surfaces. Most current knowledge on different cell types comes from studies of the spatiotemporal internal architecture of colonies developing on various media. With a few exceptions, yeast cell differentiation often concerns nongrowing, stationary-phase cells and leads to the formation of cell subpopulations differing in stress resistance, cell metabolism, respiration, ROS production, and others. These differences can affect longevity of particular subpopulations. In contrast to liquid cultures, where various cell types are dispersed within stationary-phase populations, cellular differentiation depends on the specific position of particular cells within multicellular colonies. Differentiated colonies, thus, resemble primitive multicellular organisms, in which the gradients of certain compounds and the position of cells within the structure affect cellular differentiation. In this review, we summarize and compare the properties of diverse types of differentiated chronologically aging yeast cells that have been identified in colonies growing on different media, as well as of those found in liquid cultures.

Buffering the pH of the culture medium does not extend yeast replicative lifespan

During chronological aging of budding yeast cells, the culture medium can become acidified, and this acidification limits cell survival. As a consequence, buffering the culture medium to pH 6 significantly extends chronological life span under standard conditions in synthetic medium. In this study, we assessed whether a similar process occurs during replicative aging of yeast cells. We find no evidence that buffering the pH of the culture medium to pH levels either higher or lower than the initial pH of the medium is able to significantly extend replicative lifespan. Thus, we conclude that, unlike chronological life span, replicative life span is not limited by acidification of the culture medium or by changes in the pH of the environment.

The involvement of GSH in the activation of human Sod1 linked to FALS in chronologically aged yeast cells

Mutations in Cu, Zn-superoxide dismutase (Sod1) have been associated with familial amyotrophic lateral sclerosis, an age-related disease. Because several studies suggest that oxidative stress plays a central role in neurodegeneration, we aimed to investigate the role of the antioxidant glutathione (GSH) in the activation of human A4V Sod1 during chronological aging. Transformation of wild-type and A4V hSod1 into a gsh null mutant and in its parental strain of Saccharomyces cerevisiae indicated that during aging, the number of viable cells was strongly influenced by A4V hSod1 mainly in cells lacking GSH. Activity of hSod1 increased in response to aging, although the increase observed in A4V hSod1 was almost 60% lower. Activation of hSod1 (A4V and WT) did not occur after aging, in cells lacking GSH, but could still be observed in the absence of Ccs1. Furthermore, no increase in activity could be seen in grx1 and grx2 null mutants, suggesting that glutathionylation is essential for hSod1 activation. The A4V mutation as well as the absence of GSH, reduced hSod1 activity, and increased oxidative damage after aging. In conclusion, our results point to a GSH requirement for hSod1 Ccs1-independent activation as well as for protection of hSod1 during the aging process.

Time line of redox events in aging postmitotic cells

The precise roles that oxidants play in lifespan and aging are still unknown. Here, we report the discovery that chronologically aging yeast cells undergo a sudden redox collapse, which affects over 80% of identified thiol-containing proteins. We present evidence that this redox collapse is not triggered by an increase in endogenous oxidants as would have been postulated by the free radical theory of aging. Instead it appears to be instigated by a substantial drop in cellular NADPH, which normally provides the electron source for maintaining cellular redox homeostasis. This decrease in NADPH levels occurs very early during lifespan and sets into motion a cascade that is predicted to down-regulate most cellular processes. Caloric restriction, a near-universal lifespan extending measure, increases NADPH levels and delays each facet of the cascade. Our studies reveal a time line of events leading up to the system-wide oxidation of the proteome days before cell death.DOI:http://dx.doi.org/10.7554/eLife.00306.001.

A Reduction in Age-Enhanced Gluconeogenesis Extends Lifespan

The regulation of energy metabolism, such as calorie restriction (CR), is a major determinant of cellular longevity. Although augmented gluconeogenesis is known to occur in aged yeast cells, the role of enhanced gluconeogenesis in aged cells remains undefined. Here, we show that age-enhanced gluconeogenesis is suppressed by the deletion of the tdh2 gene, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein that is involved in both glycolysis and gluconeogenesis in yeast cells. The deletion of TDH2 restores the chronological lifespan of cells with deletions of both the HST3 and HST4 genes, which encode yeast sirtuins, and represses the activation of gluconeogenesis. Furthermore, the tdh2 gene deletion can extend the replicative lifespan in a CR pathway-dependent manner. These findings demonstrate that the repression of enhanced gluconeogenesis effectively extends the cellular lifespan.